Pyrazoline B Induces Oxidative Stress Mediated Toxicity, Cell Cycle Arrest, and Caspase-Independent Apoptosis in BT-474 Human Breast Cancer Cells

Introduction

Breast cancer is one of the top five causes of death among women worldwide. In 2020, approximately 2,261,419 new breast cancer cases were reported, with 684,996 deaths worldwide.¹ The luminal B subtype accounts for 40% of all breast cancers.² Luminal B constitutes the most heterogeneous molecular subtype and has aggressive clinical and biological features.³ Luminal B breast cancer is defined by the expression of the estrogen receptor (ER+) or progesterone receptor (PR+) with or without human epidermal growth factor receptor 2 (HER2+/HER2-); it is typically graded higher, grows more quickly, and has poorer outcomes than luminal A.⁴ Moreover, the subtype of cancer also tends to grow more quickly and have a slightly worse prognosis than luminal A.⁵ Previous reports reveal a higher proportion of local recurrence and metastases in patients with luminal B breast cancer than in patients in the non-luminal group.⁶

Treatment options for luminal B breast cancer include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy, and immunotherapy.⁷ While conventional chemotherapy has shown some success, it is still plagued by issues such as poor water solubility, drug-induced toxicity, therapeutic selectivity, and drug resistance, all of which have proven to be major obstacles.^8–18 One chemotherapy drug that remains widely administered despite posing such challenges is paclitaxel. Variations in patient response rates to paclitaxel treatment have been widely documented, highlighting the need for the discovery of novel anticancer agents or therapeutic strategies.^19,20

Pyrazoline is a heterocyclic compound with two nitrogen atoms in its ring, produced by the condensation of chalcone with phenylhydrazine. These compounds have a variety of promising pharmacological activities, and present opportunities to carry out various structural modifications on the ring.^21–23 For example, in oncology, pyrazoline derivatives have been tested against numerous cancer cell lines and have demonstrated anticancer activity, such as inducing apoptosis and inhibiting cell division and proliferation.^24–28 Our previous studies report on N-phenyl pyrazoline derivatives exerting anticancer activity that have been successfully synthesized in our lab.^29–31 These compounds show biological activity in cervical cancer cells, including induction of poor survival, reduction of the HeLa’s tumorsphere size, inhibition of cancer cell growth, and reduction of CD133, the marker of cancer stem cells.^32,33 In addition, we have shown that an N-phenyl pyrazoline derivates reduces the level of epidermal growth factor receptor (EGFR), a protein that mediates the processes of cell proliferation, differentiation, survival, metastasis, and apoptosis in cervical cancer cells.^32–34 We also monitored the inhibitory activity of these compounds in T47D luminal A breast cancer cells, 4T1 and Hs578T triple-negative breast cancer (TNBC) cells, HeLa cervical cancer cells, and in WiDr colorectal cancer cells.^34,35 Furthermore, our recent study reported their capacity to improve the sensitivity of paclitaxel in TNBC cells.³⁵ The N-phenyl pyrazoline derivate is therefore a promising anticancer drug and has the potential to be combined with others to increase their efficacy. Since luminal B breast cancers tend to grow faster than luminal A, have a higher proportion of local recurrence and metastases, and have a slightly worse prognosis, we selected BT-474 cell lines representing this cancer subtype as a model to test N-phenyl pyrazoline treatment.

Materials and Methods

Materials

Chemicals were as follows: Paclitaxel (Taxol, PCXL; SRL, India), 2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI; Sigma Aldrich, USA), (methoxyphenyl)-1-phenyl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole (pyrazoline B, pyra B; laboratory of Department of Chemistry-Universitas Gadjah Mada, Indonesia) was synthesized and characterized as previously described and characterized by ¹H/¹³C Nuclear magnetic resonance (NMR), Fourier Transform IR Spectroscopy (FTIR), and Gas chromatography-mass spectrometry (GC-MS) (citation 39), Propidium Iodide (PI; Sigma Aldrich), 2-Methyl-1,4-naphthoquinone (menadione; Sigma Aldrich, USA), N-Acetyl-L-cysteine (NAC; Sigma Aldrich, USA), Pan Caspase Inhibitor (Z-VAD.fmk; Sigma Aldrich, USA), Dimethyl sulfoxide (DMSO; D8418, Sigma Aldrich, USA).

Cell Culture

Human breast cancer BT-474 cell line (ATCC, #HTB-20, USA) represent as luminal B subtype, were cultured in RPMI 1640 medium (SA R8758, Merck, Germany) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; F2442, Merck), and 1% Penicillin-Streptomycin (PS; P4458, Merck, Germany) at 37 °C and 5% CO₂ atmosphere. For toxicity treatment, cells were incubated with pyrazoline B 140 µM, pyrazoline B (140 µM) combined with paclitaxel (10 µM), pyrazoline B (140 µM) combined with Z.VAD (1 µM) for 24h. DMSO, paclitaxel, and Z-VAD.fmk were used as control. For antioxidant treatment, cells were incubated with prazoline B (140 µM), pyrazoline B (140 µM) combined with antioxidant NAC (5mM) for 24h. Stress agent menadione, the solvent DMSO, and antioxidant NAC were used as control.

Proliferation Assay

Cell proliferation was conducted using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. First, 1×10⁴ cells were plated in each well of a 96-well plate, then treated with pyrazoline B at different concentrations and incubated for 24h. Next, MTT reagent (M2128; Sigma Aldrich) was added to each well. Incubation was continued for 4h. Once the formazan crystals are formed, they are dissolved in DMSO. The quantity of formazan crystals was determined at 550 nm using a plate reader (Thermo Scientific^® Multiscan EX, Singapore).

Clonogenic Assay

For clonogenic assay, BT-474 cells were treated complete medium with pyrazoline B (140 µM), Paclitaxel (50 µM), and co-treatment cell with pyrazoline B (140 µM)-Paclitaxel (50 µM) for 24h incubation. The colony size was observed immediately after 24h incubation, using inverted microscope and was quantified using ImageJ software.³⁶

Cell Migration (Scratch) Assay

Cells were grown in 24-wells plate up to 90% confluency. After the culture medium was discarded, a scratch was made on the monolayer of cells using a p20 pipette tip. The plates were rinsed with Phosphate Buffered Saline (PBS; Gibco) and then incubated with prepared medium containing pyrazoline B (140 µM), Paclitaxel (50 µM), or pyrazoline B-Paclitaxel combination. Medium with DMSO was used as a negative control. Cells were incubated for 24 h and 72 h. The cell migration was observed using an inverted microscope (40x magnification). Cell free area were quantified using ImageJ software.³⁶

Flow Cytometry and Microscopy Assessment

For cell death analysis, PI for the membrane permeabilization and fluorescein-labeled Annexin V (Invitrogen) for the phosphatidylserine exposure were used for cytofluorometric assessment. We also showed a forward angle light scatter (FSC) channel for cell size measurement. In the gating technique, cells were selected using FSC versus side scatter (SSC) to select the cell population. Then, the cells were further gated on live cells versus cell size (PI/FSC) or live cells versus phosphatidylserine exposure (PI/Annexin V). Microscopic analysis is also conducted on cells to assess the presence of morphological changes that serve as indicators of apoptosis, such as cell rounding, membrane blebbing, nuclear condensation, and the formation of apoptotic bodies. The morphology of cells was observed using inverted microscopy (Agilent). Olympus fluorescence microscope BX51 was used to observe nuclear condensation using DAPI staining. The microscope uses a camera connected to a computer and Toupview Software (version x64, 3.7.7892) with 100x magnifications. For cell cycle analysis, the quantitation of DNA content was measured by PI fluorochrome. The cells were fixed with ethanol before staining. In the machine, cell debris was excluded by forward scatter (FSC) and side scatters (SSC). Cytofluorimetric analysis was realized using BD FACSLyric™ Flow Cytometry System (BD Biosciences) equipped with FlowJo™ software (version 10.8; BD Biosciences).

Enzyme-Linked Immunosorbent Assay (ELISA)

Briefly, 40,000 BT-474 cells (in 24 wells plate) were pre-treated with pyrazoline B for 24 h at 37 °C. Cells were then lysed in Radio-Immunoprecipitation Assay Buffer (RIPA; Sigma Aldrich). The expression levels of EGFR or VEGFR-2 proteins of the protein lysates were measured by Human Epidermal Growth Factor Receptor ELISA Kit (EGFR; Cloud-clone Corp, USA) or Human Vascular Endothelial Growth Factor Receptor 2 ELISA Kit (VEGFR-2/KDR; Elabscience, USA) according to the manufacturer’s instructions.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis

After treatment with pyrazoline B 140 µM, total RNA was isolated from the cells using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Then, mRNA expression was determined by real-time PCR using SensiFAST™ SYBR^® No-ROX One-Step Kit (Meridian Biosciences) and specific sequence primer in Agilent AriaMX PCR System (Agilent Technologies, USA). The primers were synthesized by Integrated DNA Technologies (IDT; Singapore). The nucleotide sequences (5′ to 3′) of the forward primers and reverse primers are described in Table 1. The PCR conditions were as follows: 45 °C for 10 min for reverse transcription; 95 °C for 2 min; 40 cycles of 95 °C for 5s, and 60 °C for 20s. Data were analyzed by the 2-ΔΔCT method, using the house-keeping gene β–Actin as the internal control.

Table 1 Oligonucleotide Primers

Statistical Analysis

All assays were conducted in triplicate on two or three dependent experiments, and the results were presented as the mean ± standard deviation. The ImageJ version 1.51j8 software was utilized for quantifying the data from the migration assay and colony formation assays. T For statistical significance, p-value calculations were carried out using the t-test tool in Microsoft Excel.

Molecular Docking Study and Absorption, Distribution, Metabolism and Excretion (ADME) Analysis

Protein structure was downloaded from Protein Data Bank (PDB).⁴⁴ Ligands were built using MarvinSketch (http://www.chemaxon.com) or downloaded from PDB.⁴⁴ Molecular docking was performed using UCSF Chimera⁴⁵ and Autodock Vina.^46,47 Gridbox of Flavin Adenine Dinucleotida (FAD) were set as follows: center_x = −21.00, center_y = 35.00, center_z = −24.00, size_x = 15.00, size_y = 25.00, size_z = 20.00, Gridbox of Nicotinamide Adenine Dinucleotide (NAD) were set as follows: center_x = −33.00, center_y = 31.00, center_z = −20.00, size_x = 18.00, size_y = 20.00, size_z = 20.00. Gridbox of cyp3a4 were set as follows: center_x= −15.00, center_y=−11.00, center_z=27.00, size_x=15.00, size_y=15.00, size_z=15.00. Protein and ligand visualization were performed using UCSF chimera⁴⁵ and ligplot plus.⁴⁸ ADME analysis was conducted using SWISSADME webserver, a web-based tool that is designed to evaluate the pharmacokinetics, drug-likeness, and medicinal chemistry properties of small molecules.⁴⁹

Results

Pyrazoline B Inhibits Cell Viability of BT-474 Luminal B Breast Cancer Cells

To investigate the antitumor activity of pyrazoline B, we induced BT-474 cells with various concentrations of the compound, from 7 µM to 4200 µM. The results showed a tendency to decrease cell viability, equivalent to the increasing dose of pyrazoline B (Figure 1). Cell viability was measured by MTT assay, resulting in an IC50 value of 140 µM for 24h incubation. This suggests that pyrazoline B possesses strong activity against BT-474 cells.

Figure 1 Pyrazoline B inhibits BT-474 cell growth. (A) BT-474 cells were cultured for 24h in complete medium with pyrazoline B 7, 14, 70, 140, 280, 560, 840, 1120, 1400, 2800, and 4200 µM, the medium alone (Co-1), or the solvent used to dissolve the compound (or Co-2) for 24h incubation. Cell viability was then assessed by MTT assay and expressed as a percentage of untreated cells. Values represent the mean ±SD of three independent experiments. (B) BT-474 cells were treated with pyrazoline B 140 µM or the solvent (control) for 24h. Flow cytometry was used to assay the cell cycle distribution using PI fluorescence staining. (C) Cells were treated with 140 µM pyrazoline B or solvent (control) for 24h. Total RNA was isolated from the cells using Qiagen RNeasy Mini Kit, then mRNA expression of PI3K, AKT, or Cyclin D1 was determined by real-time PCR in Agilent AriaMX PCR System. (D) Cells were treated with pyrazoline B 140 µM for 24h. The EGFR or VEGFR-2 ELISA kit detected and quantified endogenous EGFR or VEGFR-2 protein levels in the lysate sample. Two independent experiments were carried out in triplicate. Significantly different compared to the control (* p<0.05; **p < 0.01; ***p < 0.001).

Pyrazoline B Induces G0/G1 Cell Cycle Arrest and Modulates Key Signaling Pathways in BT-474 Cells

After showing the toxicity of pyrazoline B towards cancer cells, we investigated it’s impact on cell cycle progression. First, flow cytometry analysis cell cycle progression of BT-474 cells treated for 24h with 140 µM pyrazoline B showed significant accumulation in the G0/G1 phase from 54.8% in controls to 65% in cells treated by pyrazoline B, suggesting cell cycle arrest (Figure 1B). This observation prompted us to examine Cyclin D1, a critical regulator of G1-S transition. RT-qPCR analysis confirmed downregulation of Cyclin D1 at the transcriptional level (Figure 1C). We next explored upstream regulators of cell cycle control by analyzing the PI3K/AKT pathway, a known mediator of Cyclin D1 expression. Pyrazoline B treatment significantly reduced transcript levels of both PI3K and AKT (Figure 1C), indicating suppression of this crucial survival pathway. Since PI3K/AKT signaling is frequently activated by receptor tyrosine kinases, we measured the protein levels of EGFR and VEGFR-2 using ELISA. ELISA demonstrated decreased expression of both receptors in treated cells (Figure 1D). Overexpression of EGFR or vascular endothelial growth factor receptor 2 (VEGFR-2) has been reported to lead to dysregulation of crucial proteins for cell proliferation, survival, angiogenesis, and metastasis.^50,51 This coordinated inhibition of surface receptors (EGFR/VEGFR-2), their downstream effectors (PI3K/AKT), and cell cycle regulators (Cyclin D1) provides a comprehensive mechanistic explanation for pyrazoline B’s anti-proliferative effects.

Pyrazoline B Alone and in Combination with Paclitaxel Inhibits BT-474 Cell Proliferation and Migration

We next evaluated pyrazoline B’s functional effects on cancer cell behaviors. We evaluated the effect of pyrazoline B on BT-474 cell proliferation and migration. The 140 µM doses of pyrazoline B treatment suppressed the size of the colonies relative to the control (Figure 2A and B). Then, to investigate the anti-migratory effect of Pyrazoline B, we applied a wound healing assay on BT-474 cells. The result showed that using 140 µM pyrazoline B suppressed the migration of BT-474 cells (Figure 2C and D). This suppressive effect was seen for up to 72 hours of pyrazoline B incubation. These findings align with and extend our mechanistic data showing pyrazoline B’s ability to induce G0/G1 arrest, downregulate growth factor receptors, and trigger oxidative stress. As a secondary exploration of therapeutic potential, we included preliminary combination studies with paclitaxel. While not the focus of this mechanistic investigation, these assays revealed that pyrazoline B produced comparable anti-proliferative (Figure 2A and B) and anti-migratory (Figure 2C and D) effects to those of paclitaxel monotherapy and demonstrated additive effects when combined with paclitaxel (Figure 2A–D).

Figure 2 Pyrazoline B inhibits proliferation and migration of BT-474 cell line. (A and B) Pyrazoline B inhibits cell proliferation of breast cancer cells. The colony formation assay results revealed the anti-proliferative effect of pyrazoline B in BT-474 cell line. The size of BT-474 cell colonies was decreased induced by pyrazoline B. BT-474 cells were treated for 24h in a complete medium with pyrazoline B 140 µM, paclitaxel 50 µM, 140 µM pyrazoline B-50 µM Paclitaxel combination, or controls, Co-1 (solvent) or Co-2 (medium alone). The solvent control in (B) presented corresponds to Co-1 in (A). The size of colony cells was then observed under an inverted microscope. Results expressed as mean ± SD of assays done at least in triplicate. (C and D) In vitro, scratch wound healing assays showed that the cell migration ability was suppressed by pyrazoline B in a time-dependent manner. BT-474 cells were cultured for 24 and 72h in a complete medium with pyrazoline B 140 µM or solvent (control). Cell migration was then observed under an inverted microscope (40x magnification). (Cancer cell colony area and cell-free area in the scratch assay were quantified using ImageJ software). Three independent experiments were carried out in triplicate. Significantly different to control (*p < 0.05, **p < 0.01).

Pyrazoline B Induces Apoptosis in a Caspase-Independent Manner

We have previously reported that pyrazoline B induces apoptosis in cervical cancer cells. Here, we wanted to determine whether pyrazoline B causes a similar response in breast cancer cell lines, relating especially to its role in growth inhibition. BT-474 cells were treated with 140 µM pyrazoline B. Apoptotic events were evaluated by microscopy and flow cytometry analysis after 24h incubation with Pyrazoline B. Microscopic observation showed that pyrazoline B causes morphological changes typical of apoptosis, such as cell rounding, apoptotic formation, and nuclear condensation (Figure 3A and B). A decrease in cell size due to shrinkage was also observed by flow cytometry (Figure 3C). Further analysis using PI-annexin V staining showed consistent results. The number of PI-positive cells was increased after pyrazoline B treatment (PI+FSC-, 15.6%) compared to control (PI+FSC-, 2.67%) (Figure 3C). Apoptosis-related biochemical events were determined by evaluating phosphatidylserine exposure using Annexin V staining. The results showed that Annexin V-positive cells increased by 19.9% in pyrazoline B-treated PI-positive cells, compared to 4.5% in controls (Figure 3C). To explore the apoptotic mechanisms of pyrazoline-induced cell death, we next evaluated the response of cells to pyrazoline B induction in the presence of the general caspase inhibitor Z-VAD-FMK. Results showed that Z-VAD-FMK did not reduce the number of PI/Annexin V-positive cells (Figure 3C and D). This indicates that pyrazoline B induces apoptosis in a caspase-independent manner. We then analyzed the expression levels of the caspase 3 and AIF genes. Figure 3E shows that pyrazoline B induces decreased expression of the caspase 3 gene. As depicted in Figure 3E and F, pyrazoline B was found to cause a decline in caspase 3 gene expression, while simultaneously increasing the expression of AIF. These findings offer further evidence for our hypothesis that pyrazoline B triggers BT-474 cell death in caspase-independent manner.

Figure 3 Pyrazoline B induced apoptosis in BT-474 cell lines. (A) Pyrazoline B induced morphological changes typical apoptosis. Cells were treated with 140 µM pyrazoline B or control for 24h. The cell response was observed using an inverted microscope. Yellow arrow indicated round up of the cells, red arrow indicated apoptotic bodies formation, blue arrow indicated membrane blebbing. (B) We were doing fixation on treated cells or control with EtOH. Then, fixed cells were stained with nuclear staining DAPI and were observed using a fluorescence microscope. Red arrow indicated nuclear condensation. (C) Cells were treated with 140 µM pyrazoline B, 1µM Zvad.fmk+ 140 µM pyrazoline B, and 10 μM Paclitaxel+ 140 µM pyrazoline B for 24h. Cells with medium, Zvad.fmk or solvent were used as controls. Drug-induced cell death was quantified by flow cytometry of PI uptake (PI positivity), Phosphatidyl serine exposure (Annexin V positivity), and reduced cell size analysis (low forward light scatter (FSC)). Data are expressed as mean values ± SEM. (D) Further flow cytometry analysis is shown in a graph containing all cells with apoptotic features. The shrinkage cell number represents shrinkage cells with PI-positive and PI-negative (FSC-PI- and FSC-PI+). Phosphatidylserine exposure cells represent all cells with exposed phosphatidylserine in PI-positive and PI-negative cells (AnV+PI− and AnV+PI+). (E and F) Relative mRNA expression of apoptosis-related genes of Caspase-3 or AIF was determined. The expression of apoptosis-related genes in BT-474 was determined using real-time PCR. The graphs indicate the relative mRNA expression in BT-474 treated with pyrazoline B for 24 h. β-actin was used as a control. Two independent experiments were carried out in triplicate. ** Significantly different compared to the control (p < 0.01).

Pyrazoline B Inhibits Cell Growth and Induces Apoptosis Mediated by Intracellular Oxidative Stress

We have previously indicated that pyrazoline B has a good affinity with enzymes that regulate oxidative stress, such as P450 and mitochondrial apoptosis-inducing factor (AIF). This cellular stress is often associated with inducing apoptosis or inhibiting cell growth. In this study, we evaluated the involvement of pyrazoline B in oxidative stress regulation in BT-474 cells. Incubation of cells with 140 µM pyrazoline B or 10 μM menadione (positive control) for 24h resulted in a significant decrease in cell viability. The addition of extracellular antioxidants (NAC) to pyrazoline B-treated cells abolished cell viability (Figure 4A). In addition, the occurrence of oxidative stress was confirmed by increased expression of the (Hypoxia Inducing Factor-1 alpha) HIF1α gene in cells treated with pyrazoline B (Figure 4B).

Figure 4 Pyrazoline B induced oxidative stress in BT-474 cells. (A) Cells were treated with 140 µM pyrazoline B for 24h in the absence or presence of exogenous antioxidant NAC (5mM). Percentage of cell viability was determined by MTT assay. * Significantly different compared to the control (p < 0.05). #Significantly different compared to 10 ug/mL pyrazoline B (p < 0.05). (B) Cells were treated with 140 µM pyrazoline B or solvent (control) for 24h. Total RNA was isolated from the cells using Qiagen RNeasy Mini Kit, then mRNA expression of HIF1α was determined by real-time PCR in Agilent AriaMX PCR System. Two independent experiments were carried out in triplicate. ** Significantly different compared to the control (p < 0.01).

We then performed an in silico study to look at the affinity of pyrazoline B for AIF, a mitochondrial protein reported to mediate lethal redox stress and contribute to caspase-independent cell death. The results show that pyrazoline B had greater affinity than the positive control menadione, and slightly lower than another control, thiodione (Table 2). These control compounds were reported by in vitro and in vivo studies to induce caspase-independent cancer cell death.^52,53

Table 2 Affinity Energy of Ligands on AIF

The visual interaction of pyrazoline B and AIF on FAD and NAD binding sites is presented in Figure 5A and B, respectively. More detailed interaction in Figure 5C shows that pyrazoline B forms two hydrogen bonds with Arg284 and Glu452 on the FAD active site of AIF, the same contact residues as AIF-FAD and AIF-thiodione interactions. This is interesting, considering that thiodione is a menadione-conjugate compound reported to be mediated by AIF to play its lethal function in a caspase-independent manner.^52,53 In general, all ligands tended towards higher affinity for FAD than NAD binding sites, consistent with previous reports.⁵⁴ Pyrazoline B interacts using van der Waals bonds on the NAD binding domain of AIF (Figure 5D). The binding of pyrazoline B at the NAD binding site of AIF confirms the role of AIF in facilitating lethal redox cycling, resulting in antioxidant depletion observed in this study.

Figure 5 Molecular docking of pyrazoline B on AIF. (A) Visual interaction of pyrazoline B and AIF on the FAD binding site and (B) on the NAD binding site. FAD (red), pyrazoline B (green), menadione (yellow) and thiodione (blue) visualization on AIF. (C) Contact residues of pyrazoline B on the FAD binding domain of AIF and (D) on the NAD binding domain of AIF.

Cheminformatic Analysis to Predict Bioavailability of Pyrazoline B

CYP3A4 is a protein present in the small intestine and liver as a barrier against xenobiotics. CYP3A4 substrates show low bioavailability, as such compounds undergo metabolism in the small intestine or liver.⁵⁵ Paclitaxel has been known to have low oral bioavailability due to its metabolism by Cyp3a4.⁵⁶ Our in silico analysis also revealed that paclitaxel has a strong affinity against CYP3A4 (−10.1 kcal/mol) (Table 3) involving Arg105 and Arg372 via hydrogen bonding (Figures 6B and 7B). This affinity is higher when compared to erythromycin against CYP3A4 (−8.8 kcal/mol), which involves one hydrogen bond (Ser119) (Figures 6A and 7A). On the other hand, pyrazoline B exerts low affinity toward CYP3A4 (−7.8 kcal/mol) (Table 3) without hydrogen binding (Figures 6C and 7C). Furthermore, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis revealed that pyrazoline B (Log S ESOL −5.33) has a higher solubility than paclitaxel (Log S ESOL −6.66) (Figure 8). Therefore, pyrazoline B might show higher oral bioavailability than paclitaxel in future in vivo and clinical trials, since the compound does not appear to be a substrate of CYP3A4 and has a higher solubility.

Table 3 Binding Affinity of Erythromycine (Native Ligand, Pyrazoline B and Paclitaxel Against cyp3a4

Figure 6 Molecular docking result of Paclitaxel and pyrazoline B on Cyp3a4 (grey), visualized in the presence of heme fragment (grey stick). (A) Interaction of Cyp3a4 (grey) and erythromycin (yellow), (B) Interaction of Cyp3a4 (grey) and paclitaxel (red), (C) Interaction of Cyp3a4 (grey) and pyrazoline B (green).

Figure 7 Binding visualization of paclitaxel and pyrazoline B on Cyp3a4. (A) Contact residues of Cyp3a4 on Eryhthromycin’s interaction (native ligand), (B) Contact residues of Cyp3a4 on paclitaxel’s interaction, and (C) Contact residues of Cyp3a4 on pyrazoline B’s interaction.

Figure 8 Drug-likeness prediction of (A) pyrazoline B and (B) paclitaxel.

Discussion

Although chemotherapy is widely regarded as a promising strategy for fighting cancer, challenges such as poor bioavailability and low efficacy necessitate an intensive search for substances that can inhibit cell growth and induce cell death. In the present study, we demonstrate the effectiveness of pyrazoline B against BT-474 luminal B breast cancer cells, a finding that has not been reported before. Our results expand the range of known targets for the anticancer activity of pyrazoline B, having previously reported its toxicity towards T47D luminal A breast cancer cells, 4T1 TNBC mouse cells, Hs578T TNBC cells, HeLa cervical cancer cells, and WiDr colorectal cancer cells.^34,35 Key findings include induction of G0/G1 cell cycle arrest, inhibition of cell migration and colony formation, and downregulation of critical signaling pathways. Pyrazoline B’s ability to downregulate EGFR is particularly noteworthy, as cancer cells are more sensitive to EGFR downregulation than to direct inhibition of kinase activity.⁵⁷ This aligns gains additional relevance from recent evidence that EGFR inhibitors are effective in treating various types of cancer, including breast cancer;⁵⁸ yet, EGFR’s kinase-independent (KID) pro-survival function often drives therapeutic resistance.^59–61 While our in silico study suggests pyrazoline B-EGFR affinity,⁶² future studies should clarify whether pyrazoline B affects the kinase-dependent or -independent activity of EGFR through enzyme inhibition assays.

The observed VEGFR-2 protein regulation adds clinical relevance, given VEGFR-2’s established role in angiogenesis protein⁶³ and its Food and Drug Administration (FDA)-approved inhibitor for multiple types of cancers including, colorectal cancer, renal cell carcinoma, gastrointestinal stromal tumor, differentiated thyroid carcinoma, idiopathic pulmonary fibrosis, hepatocellular carcinoma, soft tissue sarcoma, and progressive neuroendocrine tumors.^58,64–66 Although VEGFR-2 inhibitors are still being explored for their potential in treating breast cancer, overexpression of the VEGFR-2 protein has been detected in early and metastatic stages of primary breast cancer.^64–66 Moreover, the overexpression of EGFR and VEGFR-2 has been linked to drug resistance and poor prognosis.^67,68 The coordinated suppression of EGFR/VEGFR-2 proteins and their downstream PI3K, AKT, and Cyclin D1, suggest that pyrazoline B may disrupt critical crosstalk between these pathways,^69,70 potentially improving clinical outcomes for breast cancer patients.^69–72 These findings represent an interesting strategy, as many anticancer drugs have been clinically approved for EGFR and VEGFR-2 activity, such as lapatinib, sunitinib, erlotinib, and sorafenib.^65,73

Inducing cancer cell death is believed to prevent recurrence and metastasis.⁷⁴ Currently, chemotherapy drugs including daunorubicin, doxorubicin, and mitomycin C are known to induce cell death.^74–76 Many clinical trials today focus on inducing cell death as a means of treating cancer.⁷⁴ Therefore, drugs that can induce cell death simultaneously with growth/proliferation inhibition are of great interest for fighting cancer cells with a dual action. This study demonstrates that pyrazoline B can induce cell death in BT-474 cells. Microscopic and flow cytometry analysis revealed that pyrazoline B-treated cancer cells undergo apoptosis, characterized by rounding of cells, cell size reduction, apoptotic body formation, and increased number of PI-Annexin V-positive cells compared to controls. We also analyzed caspase dependency in this process. The addition of the general caspase inhibitor Z-VAD-FMK did not restore cell viability, confirming that cells underwent apoptosis in a caspase-independent manner. Caspase independence was also demonstrated through the downregulation of the downstream caspase 3 and the upregulation AIF target genes. While caspase 3 downregulation might superficially suggest reduced apoptotic capacity, the concomitant AIF activation and maintained cell death confirm engagement of alternative termination pathways. This “apoptosis bypass” phenomenon has been documented with other redox-active compounds^53,77,78 and may represent an advantage for overcoming caspase-dependent resistance mechanisms. To further our understanding of this pathway, additional studies should be conducted to identify key proteins; this could involve knockdown experiments of AIF or Endonuclease G (EndoG), a protein involved in a caspase-independent mode of apoptosis. These results align with several studies evaluating drug candidates with dual actions, both inducing cell death and inhibiting EGFR/VEGFR-2.^72,79 Pyrazoline derivatives have also been previously reported to exhibit these two actions.⁸⁰

Pyrazoline B-induced intrinsic pathway apoptosis then led us to evaluate the involvement of oxidative stress in this situation. Intrinsic pathways characterized by mitochondrial involvement may be associated with redox cycling or thiol depletion, resulting in the collapse of antioxidant defenses.^53,81 It is worth noting that oxidative stress followed by apoptosis is a common feature of chemotherapeutic agents.⁸² Our study demonstrates that the cytotoxic activity of pyrazoline B was completely abolished by cell supplementation with the antioxidant N-acetyl cysteine. Pyrazoline B also provoked a decrease in cell viability, as shown in comparison with menadione, a stress agent used as a positive control. The increased expression of the HIF-1α gene also confirmed a hypoxic condition. Our findings indicate that the apoptotic pathway and oxidative stress are linked to the inhibition of cell growth and migration. This is consistent with other studies that link the activity of pyrazoline derivatives to the production of reactive oxygen species (ROS), leading to decreased cell proliferation and survival, and inducing cell death.^83,84 Together, these findings indicate that pyrazoline B is an excellent anticancer agent against BT-474 cell lines since it inhibits cell growth and proliferation, induces apoptosis, and induces oxidative stress. Since there are two kinds of luminal B cells (Her2+ and Her2-)⁸⁵ we suggest that future research explore the anticancer activity of pyrazoline B on another subtype of luminal B cancer cell lines. Additionally, future research should include genetic manipulation, such as knockdown studies, to identify the key proteins that interact with pyrazoline B directly.

Considering the toxicity potential of pyrazoline B, we conducted a simulation of its interaction with the CYP3A4 protein, part of the barrier against xenobiotics in the small intestine and liver. Our results indicated a CYP3A4 preference for metabolizing paclitaxel over pyrazoline B, leading to better bioavailability of pyrazoline B. This may be attributable to the structural properties of pyrazoline B, which has a higher solubility than paclitaxel. Structural components of paclitaxel, including its benzoyl group on the amide side chain and its acetyl group, contribute to its low water solubility and have been reported to cause adverse effects in vivo.⁸⁶ These in silico predictions, while requiring experimental validation, provide mechanistic context for pyrazoline B’s observed effects and prioritize targets for future investigation. This study establishes a foundation for subsequent experimental validation, which we are currently pursuing through AIF knockout/knockdown studies, measurement of redox cycling in purified systems, and in vivo pharmacokinetic analyses.

Anticancer drug exploration against luminal B cancer is a matter of great importance. This subtype of cancer exhibits significant molecular diversity, including variations in clinical behavior and treatment response.⁸⁷ The presented results demonstrate that pyrazoline B exerts multi-faceted anticancer activity against BT-474 luminal B cells through several distinct mechanisms: inducing of cell death, inhibition of proliferation and migration, and downregulation of EGFR/VEGFR-2 signaling pathways under hypoxic conditions. Interestingly, although not statistically significant, our preliminary data suggest enhanced anti-migratory and anti-proliferative effects when pyrazoline B is combined with paclitaxel than a single regimen.

At the molecular level, pyrazoline B uniquely induces G0/G1 cell cycle arrest, contrasting with paclitaxel’s established G2/M phase blockade.^88,89 This differential cell cycle targeting may underlie their observed synergy- a novel finding that, to our knowledge, has not been previously reported. The mechanistic basis for this synergy warrants further investigation, particularly in light of existing combination therapies. For example, while metformin-paclitaxel combination therapy leads to G2 arrest, metformin alone causes cell accumulation at the G1 phase.⁹⁰ In contrast, co-treatment of ramucirumab and paclitaxel provokes cell cycle arrest at the G0/G1 phase, while ramucirumab alone provokes G0/G1 phase arrest and is ineffective in inhibiting the G2/M phase. Together with paclitaxel, ramucirumab significantly decreased the expression of important complexes for the G2/M phase, such as cdc25A, cdc2, and Cyclin B1.⁹¹ These precedents suggest pyrazoline B might similarly influence cyclin D proteins or G2/M regulators when combined with paclitaxel, potentially explaining our observed effects on cell proliferation.

Thus, further research is necessary focusing on monitoring the effects of pyrazoline B-paclitaxel combination therapy on the cell cycle, including investigating the expression of essential proteins in the G0/G1 or G2/M phases and P13K/Akt pathway. Again, we strongly suggest the study of other luminal B cell lines to test pyrazoline B’s wider potency. In addition, further research to monitor the effect of pyrazoline B on potentiating paclitaxel activity in inducing apoptosis of cancer cells would be of interest, given that researchers have reported compounds with dual activity in potentiating paclitaxel.^92,93

The differential sensitivity across breast cancer subtypes is particularly interesting. Pyrazoline B works demonstrates about 4 times more potent against luminal A (T47D; IC50 37.78 µM) and 7.5-fold greater potency in TNBC cells (Hs578T; IC50 18.62 µM)³⁴ compared to our luminal B model. This is not surprising when we consider how these cancers behave differently. While luminal cancers typically depend on hormonal signaling pathways, TNBC cells lack these receptors and may be more vulnerable to pyrazoline B’s oxidative stress mechanisms. The intermediate sensitivity in BT-474 cells (HER2+/ER+) suggests that HER2 amplification and growth factor receptor overexpression may activate compensatory survival pathways that require higher drug concentrations.

These findings contribute to growing efforts to enhance the efficacy of paclitaxel through combining it with new drug candidates^86,92,94 by targeting multiple cancer hallmarks simultaneously. Such approaches may improve therapeutic outcomes while reducing dose-related toxicity.^95,96 Notably, even at higher concentrations, pyrazoline B maintains selectivity over Vero normal kidney cells (IC50 = 489.18 µM)³⁴ reveal a favorable 3.5-fold selectivity window for cancer cells, suggesting potential clinical applicability and its synergy with paclitaxel may offset the limitations of monotherapy potency. Future studies should prioritize comparative analyses across subtypes under identical experimental conditions to optimize subtype-specific dosing strategies, in vivo validation of combination efficacy, and comprehensive toxicity profiling to advance this compound toward clinical application.

Conclusion

This study shows that pyrazoline B exerts multi-modal anticancer activity against BT-474 luminal B breast cancer through inducing G0/G1 phase cell cycle arrest, downregulating EGFR and VEGFR-2 proteins, inhibiting PI3K/AKT/Cyclin D1 signaling pathway, inducing caspase-independent apoptosis via oxidative stress, and inhibiting cell proliferation and migration. Our preliminary combination screening revealed enhanced anti-proliferative and anti-migratory effects when pyrazoline B was paired with paclitaxel, suggesting potential therapeutic synergy. Complementing these findings, in silico analyses indicate pyrazoline B possesses favorable drug-like properties, including superior solubility and bioavailability compared to paclitaxel. Together, our data reveal the potential of pyrazoline B as an anticancer candidate. Further experiments are necessary, for example, to establish the synergy index of these combination regimens in in vitro and in vivo studies.